A Highly Multiplexed, Multiomic 3D Mouse Brain Map Using MALDI-IHC

使用 MALDI-IHC 绘制高度多重、多组学 3D 小鼠脑图

• 批准号: 10603396
• 负责人: Mark Lim
• 金额: $ 108.41万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603396
• 关键词: 3-Dimensional; Agar; Antibodies; BRAIN initiative; Biological Markers; Brain; Brain imaging; Collaborations; Computer software; Data Analyses; Data Set; Development; Disease; Fluorescence; Freezing; Goals; Hospitals; Hour; Human; Image; Image Analysis; Imaging Device; Imaging technology; Immunohistochemistry; Individual; Label; Lectin; Length; Link; Maps; Metals; Methods; Modality; Molecular; Mus; Neurodegenerative Disorders; Neurosciences; Pattern; Phase; Play; Polysaccharides; Procedures; Production; Proteins; Proteomics; Resolution; Scanning; Slice; Spatial Distribution; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Techniques; Technology; Tissue imaging; Tissues; United States National Institutes of Health; Validation; Visualization; Woman; base; brain dysfunction; brain tissue; commercialization; denoising; fluorescence imaging; glycosylation; instrumentation; mass spectrometric imaging; medical schools; metabolomics; multimodality; multiple omics; neuronal circuitry; novel; preservation; protein expression; reconstruction; small molecule; tool

Summary/Abstract A central goal of the NIH Brain Initiative is to develop new imaging tools sufficiently powerful to spatially map at high resolution the neuronal circuitry and underlying molecular composition of the brain. While cutting edge imaging tools and related labeling techniques have been developed, it is still a major challenge to map the spatial distribution at different length scales of the thousands of biomolecules, including expressed proteins, which play key roles in brain function. The goal of this Phase II project is to evaluate the ability of a new tissue imaging technology developed by AmberGen, termed MALDI-IHC, to rapidly create a highly multiplexed, multiomic and multimodal 3D molecular map of the mouse brain. The development of MALDI-IHC for whole brain imaging will provide neuroscientists with an important new tool for exploring the underlying molecular basis of brain function and neurodegenerative disorders. MALDI-IHC is based on the use of novel photocleavable mass-tags (PC-MTs) developed by AmberGen which when linked to antibody or lectin probes enable targeted biomolecules to be identified in the mass spectrometric image. This approach significantly exceeds the multiplex capability of fluorescence immunohistochemistry (IHC) and previous cleavable mass-tag based methods which are generally limited to 5 biomarkers or require extensive cycling procedures. It also exceeds the capability of metal-tagged antibody techniques such as IMC and MIBI which can probe small mm2 regions at subcellular resolution but are limited to approximately 40 antibody probes and require several days to scan a whole tissue section. In contrast, MALDI-IHC can image an entire mouse brain FFPE section for over 100 targeted proteins at 40 µm resolution in less than one hour. The ability of MALDI-IHC to perform label-free, untargeted small molecule mass spectrometric imaging (MSI), fluorescence imaging using unique dual-labeled fluorescent-PC-MT probes and high-plex imaging of intact expressed proteins including glycosylation patterns on the same tissue section greatly extends the power of this approach. During Phase I, we demonstrated the feasibility of this combined approach on mouse brain FF and FFPE tissue specimens. During Phase II, we will develop methods using MALDI-IHC to reconstruct whole mouse brain protein expression maps at 40 µm voxel resolution. FFPE sagittal and coronal mouse brain tissue slices from mouse brain will be probed by MALDI-IHC using a panel of 50 NeuroMab PC-MT antibodies and 25 PC-MT lectins. Validation of individual PC-MT probes will be performed by comparing MALDI-IHC and fluorescence IHC images. A 3D tri-modal map of the mouse brain merging both metabolites and expressed proteins will also be reconstructed based on a demonstrated workflow that involves MSI of unlabeled small molecules from successive FF specimens, IHC staining with a 75-plex panel of PC-MT probes including some dual-labeled PC-MT antibodies, and fluorescence imaging followed by MSI of the PC-MTs. Reconstruction of 3D maps, visualization and image analysis will be performed using Bruker SCiLS™ software. Commercialization of MALDI-IHC technology will be accelerated by a close collaboration with Bruker Daltonics, the market leader of MALDI-MSI instrumentation.

摘要/摘要 NIH脑倡议的一个核心目标是开发足够强大的新成像工具，可以在空间上绘制 高分辨率大脑的神经元回路和基础分子组成。同时尖端 成像工具和相关的标签技术已经开发出来，绘制空间仍然是一个主要挑战 在不同的长度尺度上分布的数千种生物分子，包括表达的蛋白质， 在大脑功能中的关键作用。该阶段项目的目标是评估新组织成像的能力 由Ambergen开发的技术，称为MALDI-IHC，以迅速创建高度多重的，多瘤和 小鼠脑的多模式3D分子图。 MALDI-IHC用于全脑成像的开发将 为神经科学家提供一个重要的新工具，用于探索大脑功能的基本分子基础 和神经退行性疾病。 MALDI-IHC基于使用新型光电标记（PC-MTS）的使用 由Ambergen开发的，当与抗体或讲座问题相关时，可以使靶向生物分子成为 在质谱图像中识别。这种方法大大超过了 荧光免疫组织化学（IHC）和先前的基于可切合的质量标签方法通常是 限制为5个生物标志物或需要广泛的骑自行车程序。它也超出了金属标签的能力 抗体技术，例如IMC和MIBI，可以在亚细胞分辨率下探测小的MM2区域 仅限于大约40个抗体问题，需要几天来扫描整个组织部分。相比之下， MALDI-IHC可以以40 µm分辨率在40 µm分辨率的100多个靶向蛋白的整个小鼠脑FFPE截面中成像 不到一个小时。 MALDI-IHC执行无标签，未靶向的小分子质谱的能力 成像（MSI），使用独特的双标记荧光-PC-MT探针和高型成像的荧光成像 完整表达的蛋白质包括在同一组织部分上的糖基化模式，大大扩展了功率 这种方法。在第一阶段，我们证明了这种联合方法对小鼠脑FF的可行性 和FFPE组织标本。在第二阶段，我们将使用MALDI-IHC开发方法来重建整体 小鼠脑蛋白表达图在40 µM体素分辨率下。 FFPE矢状和冠状小鼠脑组织切片 MALDI-IHC将使用50个神经元PC-MT抗体和25 pc-mt的面板探测从小鼠脑中探测的。 凝集素。通过比较MALDI-IHC和荧光IHC，将对单个PC-MT问题进行验证 图像。融合两种代谢物和表达蛋白的小鼠脑的3D三模式图也将是 基于证明的工作流程重建，该工作流程涉及成功的未标记小分子的MSI FF标本，IHC染色，具有75个PC-MT问题，包括一些双标记的PC-MT抗体， 和荧光成像，然后是PC-MTS的MSI。重建3D地图，可视化和图像 分析将使用Bruker SCILS™软件进行。 MALDI-IHC技术的商业化将是 与Maldi-MSI仪器市场领导者Bruker Daltonics进行了密切合作，加速了。